Resumo

Currently licensed vaccines against infections caused by Streptococcus pneumoniae are based on the induction of antibodies against capsular polysaccharides. The conjugate vaccines are very efficient in the protection against invasive disease caused by vaccine serotypes, but the widespread use of the 7-valent conjugate vaccine has led to an increase in disease caused by non-vaccine serotypes. Alternative vaccines are thus still necessary. S. pneumoniae has proteins that are attached to the surface of the bacteria through affinity with the choline residues from teichoic and lipoteichoic acids of the cell wall and cell membrane, respectively. When grown in chemically defined medium without choline and with ethanolamine (CDM-ET), these choline-binding proteins (CBPs) are released to the culture supernatant. CBPs are an interesting alternative for the development of a cost-effective vaccine with broad coverage and, in fact, immunization with CBPs was previously shown to protect mice against different lethal challenges with pneumococci. Since PspA (Pneumococcal surface protein A) is an antigen that shows variability between strains and it is believed to be the most important protective component among the different CBPs, we aimed at constructing an Rx1 strain expressing PspA from clade 4 (PspA4). We have previously shown that PspA4 induces antibodies that recognize most PspA variants. We have first used the Janus Cassette to knockout pspA, generating Rx1-ΔpspA, and this isolate was then transformed with the pspA4 gene and flanking regions from strain St255/00, generating Rx1-pspA4. SDS-PAGE analysis showed the presence of several bands in the supernatant from Rx1-pspA4 grown in CDM-ET, but mass spectrometry (MS) analysis showed that the most prominent bands were not CBPs. We have thus grown Rx1-pspA4 in Todd-Hewitt medium containing 0,5% yeast extract and washed the cells in 2% choline chloride (CC) as an alternative to recover CBPs. SDS-PAGE analysis of the proteins recovered by the CC wash showed few bands, which were identified as PspA and PspC in MS-analysis. Immunization of mice with the proteins recovered from the CC wash of Rx1-pspA4 without adjuvant led to a significantly higher protection against an intranasal lethal challenge with S. pneumoniae. Supported by FAPESP and CNPq (Brazil).